BACKGROUND Congenital disorders of glycosylation (CDG) represent 1 of the largest groups of metabolic disorders with >130 subtypes identified to date. The majority of CDG subtypes are disorders of N-linked glycosylation, in which carbohydrate residues, namely, N-glycans, are posttranslationally linked to asparagine molecules in peptides. To improve the diagnostic capability for CDG, we developed and validated a plasma N-glycan assay using flow injection–electrospray ionization–quadrupole time-of-flight mass spectrometry. METHODS After PNGase F digestion of plasma glycoproteins, N-glycans were linked to a quinolone using a transient amine group at the reducing end, isolated by a hydrophilic interaction chromatography column, and then identified by accurate mass and quantified using a stable isotope-labeled glycopeptide as the internal standard. RESULTS This assay differed from other N-glycan profiling methods because it was free of any contamination from circulating free glycans and was semiquantitative. The low end of the detection range tested was at 63 nmol/L for disialo-biantennary N-glycan. The majority of N-glycans in normal plasma had <1% abundance. Abnormal N-glycan profiles from 19 patients with known diagnoses of 11 different CDG subtypes were generated, some of which had previously been reported to have normal N-linked protein glycosylation by carbohydrate-deficient transferrin analysis. CONCLUSIONS The clinical specificity and sensitivity of N-glycan analysis was much improved with this method. Additional CDGs can be diagnosed that would be missed by carbohydrate-deficient transferrin analysis. The assay provides novel biomarkers with diagnostic and potentially therapeutic significance.

Deficiency of carnitine palmitoyltransferase 1A (CPT1A) results in impaired hepatic long-chain fatty acid oxidation and ketogenesis. We have previously described a patient with a severe CPT1A phenotype who is homozygous for the nonsense mutation 478 C > T (R160X). It has been known for some time that gentamicin can promote readthrough of nonsense codons. Recently, a new compound (PTC124) with less clinical toxicity than gentamicin has been indicated as a therapy for patients with nonsense mutations for multiple genetic diseases. The study is designed to investigate whether PTC124 can promote readthrough of the R160X CPT1A mutation and increase normal sized CPT1 protein expression and activity in the patient's skin fibroblasts. Our study demonstrated that after both PTC 124 and gentamicin treatment, there was an increase in CPT1 activity in patient fibroblasts to levels that are similar to that of the mild Inuit P479L variant. Our results provide additional evidence for proof of principle that PTC124 is a potential therapeutic agent for treating patients with any genetic condition that results from a nonsense mutation.

Background Measurement of amino acids in dried blood spots has been extensively used for the detection of newborns with various inborn errors of amino acid metabolism including phenylketonuria (PKU) and maple syrup urine disease (MSUD). Whereas blood spot amino acid measurement has been invaluable for initial diagnosis, the relative insensitivity of blood spot measurement has found limited use in lifelong monitoring of patients with these disorders. The work described here outlines the evaluation of blood spot amino acid analysis using ultra-performance liquid chromatography (UPLC©) for use in follow-up testing. Methods Dried blood spot amino acids were derivatized with a proprietary AccQTag® reagent and separated using UPLC. Plasma amino acids from dried bloods spots were obtained from 318 patient samples and compared to corresponding plasma samples measured using the same UPLC method. Results Dried blood spot amino acid concentrations were highly correlated but negatively biased vs plasma concentrations. Interassay imprecision studies using UPLC demonstrated a %CV for phenylalanine of 4.81%–16.07%, tyrosine 5.62%–20.16%, valine 4.23%–15.46%, leucine 8.3%–15.3%, and isoleucine 4.25%–16.80%. Intraassay imprecision studies using UPLC demonstrated a %CV for phenylalanine of 0.42%–3.4%, tyrosine 1.6%–7.85%, valine 0.14%–1.84%, leucine 0.28%–2.01%, and isoleucine 0.6%–2.65%. Blood spot amino acid concentrations were stable for at least 3 days at temperatures up to 65 °C. Conclusions This UPLC-based method can reliably measure clinically significant amino acids in dried blood spots.

Medium- and short-chain L-3-hydroxyacyl-coenzyme A dehydrogenase (M/SCHAD, SCHAD) deficiency is a mitochondrial fatty acid oxidation disorder (FAOD). This enzyme catalyzes the penultimate step in fatty acid oxidation, the NAD+ dependent conversion of L-3-hydroxyacyl-CoA to 3-ketoacyl-CoA for medium- and short-chain acyl-CoA intermediates (C4-C12). The clinical presentations of most patients are recurrent hypoglycemia associated with hyperinsulinism. We presented four infants with C4 acyl-carnitine elevation identified by newborn screening that also showed an unusual phenotype of congenital hypotonia and gross developmental delay. Enzymatic studies confirmed the disease. Sequencing analysis of all the HADH coding exons on the four patients revealed a homozygous variant of a novel change (c.908G>T, p.Gly303Val). Western blot analysis subsequently confirmed the lack of the SCHAD protein. In addition, there is another previously reported benign variant (c.257T>C) identified in three infants. Therefore, we postulate that the HADH variant (c.908G>T) is indeed pathogenic and associated with a severe phenotype as evidenced by the cases described herein. Population screening for the c.908G>T mutation suggests this mutation to be common among Puerto Ricans. We recommend that SCHAD deficiency is included as part of the differential diagnosis of all infants with congenital hypotonia.

Depth-profile analyses of insulating materials have been frequently carried out with an ion gun. The use of an ion gun generally results in preferential sputtering and poor depth resolution. Uniform sputtering of nonconductive powders can be achieved using a low-pressure radio-frequency plasma. The powders were dispersed in a conductive medium to enhance the charge dissipation. The technique was tested with many nonconductive powders including latex spheres, Y2O3, Zn2SiO4, BaSi2O5 and ZrO2. Preferential sputtering caused by charging was not observed in any of the samples studied. In the case of ZnS, preferential sputtering was detected but it was the result of multicrystallite orientations existing in the sample.